Transcription factors represent a group of molecules within the cell that function to connect the pathway from extracellular signal to intracellular response. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to DNA and activate the transcription of their target genes. One such group of transcription factors is the Rel/NF-kappa-B family. These transcriptional activators are found in many tissues and are localized in the cytosol as hetero- or homodimers with other members of the family and remain inactive due to the association of the dimer with an inhibitory protein. Rel/NF-kappa-B proteins have been shown to be involved in a diverse set of signaling pathways involving stress, apoptosis, cancer, growth, infection, and inflammation.
The p65 subunit of NF-kappa-B (also known as RelA, NFKB3 and NF-kappa-B p65 subunit) is a member of the Rel/NF-kappa-B family of transcription factors which includes p50, cRel, p52 and RelB. NF-kappa-B p65 subunit was first isolated from Jurkat T cells using a probe that spanned a conserved domain to the proto-oncogene cRel (Ruben et al., Science, 1991, 251, 1490-1493) and since, a naturally occurring transforming variant of the protein has been shown to exist (Narayanan et al., Science, 1992, 256, 367-370).
NF-kappa-B p65 subunit can form homo- and heterodimers and once the dimer dissociates from an inhibitor molecule it translocates to the nucleus where it affects the transcription of several target genes including NF-kappa-B family members and those of the inhibitors (Baeuerle and Henkel, Annu. Rev. Immunol., 1994, 12, 141-179). Other genes regulated by NF-kappa-B p65 subunit-containing dimers include cytokines such as Interleukin-2, Interleukin-6, Interleukin-8, and granulocyte macrophage-colony stimulating factor (GM-CSF), adhesion molecules such as vascular cells adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) and several acute phase response proteins (Ghosh et al., Annu. Rev. Immunol., 1998, 16, 225-260). It has recently been demonstrated that the ability of NF-kappa-B p65 subunit to activate transcription is regulated by Protein kinase A, which modulates the interaction of NF-kappa-B p65 subunit with CBP/p300 (Zhong et al., Mol. Cell, 1998, 1, 661-671).
Manifestations of altered NF-kappa-B p65 subunit regulation appear in disease states and NF-kappa-B p65 subunit transcriptional activation has been implicated in Hodgkin's disease (Bargou et al., J. Clin. Invest., 1997, 100, 2961-2969), multiple sclerosis (Gveric et al., J. Neuropathol. Exp. Neurol., 1998, 57, 168-178) and atherosclerosis (Bourcier et al., J. Biol. Chem., 1997, 272, 15817-15824). NF-kappa-B p65 subunit has been shown to be overexpressed in breast cancers (Sovak et al., J. Clin. Invest., 1997, 100, 2952-2960) and down-regulated in colon cancers (Cadoret et al., Oncogene, 1997, 14, 1589-1600).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of NF-kappa-B p65 subunit. To date, strategies aimed at inhibiting and/or investigating NF-kappa-B p65 subunit function have involved the use of antibodies, dominant-negative forms of NF-kappa-B p65 subunit, antisense technology and gene knock-outs in mice.
Using anti-NF-kappa-B, p65 antibodies Sioud et al. localized NF-kappa-B p65 subunit to the nuclei of synovial tissues of patients with rheumatoid arthritis and juvenile rheumatoid arthritis suggesting a role for NF-kappa-B signaling in this disease (Sioud et al., Clin. Exp. Rheumatol., 1998, 16, 125-134).
An antisense molecule targeted to NF-kappa-B p65 subunit which prevents cellular adhesion is disclosed in U.S. Pat. No. 5,591,840 (Narayanan and Rosen, 1997). This sequence is targeted to the 5' end of the mRNA encoding human NF-kappa-B p65 subunit and extends to 3 nucleotides upstream of the initiation codon. This same antisense oligonucleotide, as a phosphorothioate, has also been shown to suppress the synthesis of uPA, an enzyme that cleaves plasminogen in ovarian cancer cells (Reuning et al., Nucleic Acids Res., 1995, 23, 3887-3893). It has also been used to abolish NF-kappa-B p65 subunit expression, to elucidate the role of NF-kappa-B p65 subunit in resting and stimulated granulocytes (Sokoloski et al., Blood, 1993, 82, 625-632) and to further illustrate the role of NF-kappa-B p65 subunit in inhibition of cell adhesion (Khaled et al., Nucleic Acids Res., 1996, 24, 737-745). Crofford et al. used the same oligonucleotide (citing Sokoloski (Sokoloski et al., Blood, 1993, 82, 625-632), though the oligonucleotide is shown with an additional G in the 13.sup.th position) to reduce the expression of COX-2, an enzyme whose expression is induced by proinflammatory stimuli in synoviocytes (Crofford et al., Arthritis Rheum., 1997, 40, 226-236). Binding of the p65/p50 heterodimer to the COX-2 promoter was reduced in the antisense-treated cells. In addition, a longer form of this antisense molecule was used to show that NF-kappa-B p65 subunit antisense exerted antigrowth effects in many transformed cells (Sharma and Narayanan, Anticancer Res., 1996, 16, 589-596). Antisense oligonucleotides to NF-kappa-B p65 subunit are also disclosed in WO 97/47325 and WO 95/35032 (Kitajima and Nerenberg, 1995; Neurath et al., 1997). These are very similar to the oligonucleotide disclosed in U.S. Pat. No. 5,591,840 differing only by two and three bases, respectively. Antisense oligonucleotides to the corresponding section of the NF-kappa-B p65 subunit gene in mice have also been used to investigate NF-kappa-B p65 subunit fucntion (Benimetskaya et al., Nucleic Acids Res., 1997, 25, 2648-2656; Hamdane et al., Oncogene, 1997, 15, 2267-2275; Higgins et al., Proceedings of the National Academy of Science USA, 1993, 90, 9901-9905; Ivanov et al., Oncogene, 1997, 14, 2455-2464; Maltese et al., Nucleic Acids Res., 1995, 23, 1146-1151; Narayanan et al., Molecular and Cellular Biology, 1993, 13, 3802-3810; Perez et al., Molecular and Cellular Biology, 1994, 14, 5326-5332).
Mice lacking the NF-kappa-B p65 subunit gene died in utero at 15 days of gestation and presented with degeneration of the liver caused by excessive apoptosis. However, progenitor cells from these animals gave rise to a normal T cell repertoire (Beg et al., Nature, 1995, 376, 167-170). Alternatively, studies involving mice overexpressing NF-kappa-B p65 subunit in the thymus found that the major mechanism controlling NF-kappa-B activation is the cytoplasmic retention of NF-kappa-B p65 subunit by the I-kappa-B inhibitor (Perez et al., Mol. Cell Biol., 1995, 15, 3523-3530).
In light of these studies, it is still believed that there remains a need for additional agents capable of effectively inhibiting NF-kappa-B p65 subunit function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of NF-kappa-B p65 subunit expression.